Design, synthesis and use of  synthetic nucleotides comprising charge mass tags

ABSTRACT

Embodiments of the present disclosure relate generally to reporter compositions which are synthetic nucleotides that comprise nucleotides with a high charge mass moiety attached thereto via a linker molecule. The linker molecules can vary in length in part to enable the high charge mass moiety to extend out from a DNA polymerase complex so that polymerization may not be influenced.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Some embodiments of the present disclosure relate generally to syntheticnucleotides that comprise nucleotides with a charge mass reportermolecule via a long linker molecule. The linker molecules can vary inlength in part to enable the reporter moiety to extend out from the DNAPolymerase complex so that some aspects of polymerization may not beinfluenced entirely or partially.

2. Description of the Related Art

A nucleotide can be defined as a phosphate ester of a nucleoside,comprising a purine or pyrimidine base linked to ribose, or deoxyribosephosphates. The purine nucleotides having chiefly Adenine (A) or Guanine(G) as the base, the pyrimidine nucleotides Cystine (C), Thymoine (T) orUracil (U), and which are the basic repeating units in DNA and RNA(Henderson's dictionary of biological terms, 1989).

DNA is a long polymer comprising units of deoxyribose nucleotides andRNA is a polymer of ribose nucleotides. This sequence of nucleotidebases can determine individual hereditary characteristics.

The central dogma of molecular biology generally describes the normalflow of biological information: DNA can be replicated to DNA, thegenetic information in DNA can be ‘transcribed’ into mRNA, and proteinscan be translated from the information in mRNA, in a process calledtranslation, in which protein subunits (amino acids) are brought closeenough to bond, in order (as dictated by the sequence of the mRNA &therefore the DNA) by the binding of tRNA (each tRNA carries a specificamino acid dependant on it's sequence) to the mRNA.

SUMMARY OF THE INVENTION

A reporter composition is disclosed in accordance with embodiments ofthe present invention. The reporter composition comprises a nucleotideor its derivative, a linker molecule, which may be attached to thenucleotide or its derivative, and a high charge mass moiety, whichcomprises a charge mass that is sufficient to change a property of asensitive detection nanostructure operably coupled to the reportercomposition. In some embodiments, the nucleotide or its derivativepresent in the reporter composition may be selected from the groupconsisting of a deoxyribonucleotide, a ribonucleotide, a peptidenucleotide, a morpholino, a locked nucleotide, a glycol nucleotide, athreose nucleotide, any synthetic nucleotides, any isoforms thereof, andany derivatives thereof.

In some other embodiments, the linker molecule comprises a molecule ofthe following general formula, H₂N-L-NH₂, wherein L may comprise alinear or branched chain comprising an alkyl group, an oxy alkyl group,or the combination thereof. L in the linker may comprise a linear chaincomprising an alkyl group, an oxy alkyl group, or the combinationthereof and a number of the alkyl group, the oxy alkyl group, or thecombination thereof in the linear chain is 1 to 100, 1 to 75, 1 to 50, 1to 25 or 1 to 1000 in various examples. In some examples, the number ofthe alkyl group, the oxy alkyl group, or the combination thereof in thelinear chain can be more than 1000. The linker molecule and/or the highcharge mass moiety is configured not to affect nucleotide polymerizationby a polymerase and also be removable. The linker molecule can be linkedto a phosphate group, sugar group or base of the nucleotide or itsderivative.

In some embodiments, the high charge mass moiety present in the reportercomposition can be positive or negative, and further the charge mass ofmoiety can be variable depending on pH. In some examples, the highcharge mass moiety may comprise an aromatic and/or aliphatic skeleton,wherein the skeleton comprises one ore more of a tertiary amino group,an alcohol hydroxyl group, a phenolic hydroxy group, and anycombinations thereof. In some examples, the high charge mass moietycomprises one or more of the following groups, any derivatives thereof,and any combinations thereof:

In addition, the number of the foregoing groups, any derivativesthereof, and any combinations thereof present in the high charge massmoiety can be 1 to 10, 11 to 50, 51 to 100, or more than 100 in variousembodiments.

In one aspect of the invention, a reporter composition may comprise thefollowing molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

In another aspect of the invention, a reporter composition may comprisethe following molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

In still another aspect of the invention, a reporter composition maycomprise the following molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

A kit for determining a nucleotide sequence, comprising the reportercomposition comprising a nucleotide or its derivative, a linkermolecule, and a high charge mass moiety is also disclosed in accordancewith embodiments of the present invention.

A method of synthesizing the reporter composition is also disclosed. Themethod comprises: generating a first covalent linkage between thenucleotide or its derivative and a first amine group of the linker,wherein a phosphate group, a sugar or a base of the nucleotide or itsderivative is linked to the first amine group of the linker; andgenerating a second covalent linkage between a second amine group of thelinker and any functional group present in the high charge mass moiety:wherein the linker comprises at least two amine groups is also disclosedin connection with the present application. In some embodiments, thenucleotide or its derivate used in the method may comprises amonophosphate group. In some other embodiments, the nucleotide or itsderivate used in the method may be selected from the group consisting ofadenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidinemonophosphate (CMP), thymidine monophosphate (TMP), and uridinemonophosphate (UMP).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The sequencing of the human genome and the subsequent studies have sincedemonstrated the great value in knowing the sequence of a person's DNA.The information obtained by genomic DNA sequence analysis can provideinformation about an individual's relative risk of developing certaindiseases (such as breast cancer and the BRCA 1&2 genes). Furthermore,the analysis of DNA from tumors can provide information about stage andgrading.

Infectious diseases, such as those caused by viruses or bacteria alsocarry their genetic information in nucleotide polymer genomes (eitherDNA or RNA). Many of these have now been sequenced, (or enough of theirgenome sequenced to allow for a diagnostic test to be produced) and theanalysis of infectious disease genomes from clinical samples (a fieldcalled molecular diagnostics) has become one of important methods ofsensitively and specifically diagnosing disease.

Measurements of the presence or absence, as well as the abundance ofmRNA species in samples can provide information about the health statusof individuals, the disease stage, prognosis and pharmacogenetic andpharmacogenomic information. These expression arrays are fast becomingtools in the fight against complex disease and may gain in popularity asprices begin to fall.

In short, the analysis of nucleotide polymers (DNA & RNA) has becomeimportant in the clinical routine, however, cost remains a barrier towidespread global adoption. One reason for this is the complexity of theanalysis requiring expensive devices that are able to sensitivelymeasure up to four different fluorescence channels as RT-PCR experimentsprogress. The cheaper alternatives may require skilled technicians torun and interpret low-tech equipment, such as electrophoresis gels, butthis too may be expensive and a lack of skilled technicians indeveloping countries is prohibitive.

To solve this, a method of nucleotide polymer analysis that may requirecheap and easy to use devices may be required. Some embodiments of thepresent disclosure describes chemical reagents, synthetic nucleotides,that can generally be utilized in such devices. Various embodiments usedin connection with of the present disclosure describes novel syntheticnucleotides that comprises at least some standard nucleotides (or anymodifications, or isoforms), with a high negative charge mass reportermoiety attached via a linker molecule (for instance, attached to the 5′phosphate group), with the linker length of such a length so as toprotrude from a polymerase complex during polymerization, so as not tocause a significant deleterious effect on the polymerase's action.

As used in various embodiments herein, a nucleotide can be, but notlimited to, one of the following compounds, Adenine, Guanine, Cytosine,Thymine, Uracil, and Inosine as well as any modified nucleotides, anynucleotide derivatives and any degenerate base nucleotides. Somenon-limiting examples of such nucleotide may comprise adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof. Furthermore, single stranded deoxyribose nucleicacid (ssDNA) can generally be a single stranded nucleotide polymermolecule, comprising Nucleotides and double stranded deoxyribose nucleicacid (dsDNA) can generally be a double strand comprising two ssDNAmolecules linked together via, for example, hydrogen bonding, in areverse complimentary orientation.

Nucleotides can generally be synthesized through a variety of methodsboth in vitro and in vivo. This can involve salvage synthesis (there-use of parts of nucleotides in resynthesizing new nucleotides throughbreakdown and synthesis reactions in order to exchange useful parts), orthe use of protecting groups in a laboratory. In the latter case, apurified nucleoside or nucleobase can be protected to create aphosphoramidite, and can be used to obtain analogues not present innature and/or to create an oligonucleotide.

In some embodiments, nucleotide synthesis comprises the formation of anucleoside (the nitrogenous base joined to a sugar). The sugar involvedin the synthesis and structure of a nucleotide may be either ribose ordeoxyribose; in the latter case, the prefix ‘deoxy’ may be added beforethe name of the nucleoside in all cases except Uracil. A functionalgroup of phosphate can be then esterified to the sugar, creating anucleotide. The phosphate group may comprise one, two, or threephosphates, forming mono-phosphates, di-phosphates, or tri-phosphates,respectively.

Some other embodiments of the present disclosure describe the design,synthesis and use of special synthetic nucleotides comprising anucleotide and a reporter moiety, in which the reporter moiety may notact as a polymerase enzyme blocking moiety attached via a linker.

A reporter moiety or reporter composition used in various embodiments inconnection with the present inventions is a molecule or molecules thatare easily detected by a biosensor or other detection method (such as byeye) and are attached to biomolecules, or probes, or primers that detector amplify molecules of interest.

A linker molecule used in various embodiments is a polymer made up ofmore than one subunit that links a reporter molecule to a nucleotide. Anexample of a linker molecule is a Di-amine linker H₂N-L-NH₂, where Lrepresents a number of further subunits.

As such, the present disclosure should be considered to include allconfigurations that include any nucleotide or its derivative with alinker molecule attaching a reporter moiety with an overall high charge,sufficient enough to get a detectable change in a sensitive biosensorthat can detect small variations in charge mass at or near its surface.Accordingly several examples presented in this application are presentedonly for the purpose of illustration and should not be considered tolimit the scope of the invention.

In various embodiments, the synthetic nucleotides can have at least someof the following aspects:

-   -   1. The reporter moieties reports based upon its charge mass, not        enzymatic activity, fluorescence etc;    -   2. Each synthetic nucleotide may either carry the same charge        mass reporter moiety, or carry a different charge mass;    -   3. The reporter moieties may be easily cleaved; and/or    -   4. The nucleotides may be cheaply and easily mass synthesized.

There are several possible positions available for the attachment oflinkers and the reporter moieties. It is important to attach the linkerso as to not interfere with polymerization or hydrogen bonding betweenthe bases of nucleotides when hybridizing with its compliment base inanother nucleotide polymer (i.e. when two strands of reverse complimentDNA hybridize to form a double stranded DNA molecule), One possibleposition can be the phosphate linkage in the nucleotide. Furthermore, byattaching the linker to the 5′ position in the phosphate, it will blockfurther nucleotide additions as it will prevent phosphodiester bondformation.

Another possible positions available for the attachment of linkers andthe reporter moieties, is on either the sugar group or the base group.

Some other embodiments describe methods of the use in which the linkerand reporter moiety can be cleaved from the synthetic nucleotide in theiterative manner after detection. As one of the possible places toattach the linker is the 5′-phosphate end of phosphate group, which willprevent further nucleotide additions then, by cleaving the linker willtherefore remove this block and allow for further nucleotide additions.

As way of an example, there are at least two options available whichcould facilitate synthesis at the 5′-phosphate terminal:

1. Thiophosphate; and/or

2. Phosphoramidate.

The proposed linker therefore can have the following structure at leastin some embodiments:

H₂N-L-NH₂

where, L could be, but is not limited to, any linear or branched chainmolecule that is configured to link to a nucleotide as well as a highcharge mass moiety, both of which are present in a synthetic nucleotide.In some embodiments, L comprises a plurality of an alkyl group, an oxyalkyl group or the combination thereof with various lengths. In oneembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 100. In another embodiment, the numberof an alkyl group, an oxy alkyl group or the combination thereof in L is1 to 75. In still another embodiment, the number of an alkyl group, anoxy alkyl group or the combination thereof in L is 1 to 50. In stillembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 25. In some other embodiments, thenumber of an alkyl group, an oxy alkyl group and the combination thereofin L can be more than 100. While NH₂ is presented for the purpose ofillustration, this NH₂ can be substituted with any other function groupthat can be cross-linked to a nucleotide or its derivative as well as ahigh charge mass moiety, both of which are present in a syntheticnucleotide. Some illustrative examples that can be used instead of NH₂include, but not limited to, any alkyl group (e.g. C_(n)H_(2n+1),wherein n represents a positive integer number such as 1, 2, 3, andetc), any alcohol group (e.g. C_(n)H_(2n)OH, wherein n represents apositive integer number such as 1, 2, 3, and etc), any carboxyl group(e.g. COOH), any amide group (e.g. CONH), and any derivatives thereof.As the linker molecules can vary in length and chemical structure inpart to enable the reporter moiety to extend out from a nucleotidepolymerase (e.g. DNA polymerase, RNA polymerase and others) complex sothat some aspects of polymerization may not be influenced entirely orpartially

The easy access to the linkers of various lengths can be considered as abenefit in a situation where the desired length of the linker may not beknown completely or partially. This may make the optimizationexperiments easy.

The linker with the nucleotide (say Adenosine as an illustrativeexample) therefore may have the following structure at least in someembodiments. While adenosine is presented in some examples below, thisadenosine can be substituted with any other natural or syntheticnucleotide, any modifications thereof and any derivatives thereof insome other embodiments.

In some embodiments, various lengths of linkers at this position mayhave the following structures (exemplified with the Adenosine):

1. Ethylenediamine (2 Carbon Bond Length Separation)

2. Pentanediamine (5 Carbon Bond Length Separation)

3. Length Equivalent to 13 Carbon Bond Length Separation

Thus in some embodiments, the linkers thus selected can be:

1. Easily available;

2. Easy to link and cleave (please refer the probable protocols below);and/or

3. Not to interact with the polymerase and the polynucleic acid strandand/or not affect nucleotide polymerization and growth of a nascentnucleotide polymer.

The reporter moiety: As used herein a “reporter moiety” is a molecule ormolecules that are easily detected by a biosensor or other detectionmethod (such as by eye) and are attached to biomolecules, or probes, orprimers that detect or amplify molecules of interest that are normallydifficult to detect without the presence of the reporter moiety‘reporting’ on its presence. The reporter moieties can be any chargedmolecule, group of charged molecules and even many charged moleculesarranged dendritically. The reporting mode is their charge, which isdetected by sensitive charge detection biosensors, such asnanowire/nanotube FETS, nanopores and other piezoelectric biosensors. Insome embodiments, the reporter moieties can be associated with the otherproperties like the chromophoric nature for enabling their detection byUV or visible detector or the fluorescent nature making them to bedetected by the fluorimetric detection. Furthermore, the mass of thereporter moiety can be exploited using biosensors that can detect mass,such a surface Plasmon resonance biosensors and cantalivers.

The charge on the reporter: certain embodiments of the present inventiondescribe the reporter moiety to carry a large charge mass. In oneembodiment, the reporter moiety may introduce a higher charge mass tothe synthetic nucleotide than the charge mass of the nucleotide or itsderivative, which is present in the synthetic nucleotide. However, inanother embodiment, the charge mass introduced by the reporter moietycan be substantially equal to or less than the charge mass of thenucleotide or its derivative, which is present in the syntheticnucleotide. Some non-limiting and illustrative examples of a reportermoiety are provided in this specification. These examples are providedonly for the illustration purpose and therefore should not be consideredto limit the scope of the invention. The chemical structure and/ordimension (e.g. length, size, and mass of a molecule used as a reportermoiety) of a reporter moiety may not be restricted as long as thereporter moiety is configured to provide a charge mass to the syntheticnucleotide and also not to affect polymerization reaction of nucleotidespartially or entirely.

The charge on the moiety can be positive or negative. Taking intoconsideration the nature of linkage, the following provides some aspectsof the selection of charge that can be possibly used in some embodimentsof the present disclosure.

Positive charge: In some embodiments, the large number of positivecharges can generally be induced on the reporter moiety through theincorporation of tertiary amino groups on the aromatic or aliphaticskeleton. In such embodiments, in turn in the acidic pH (less than 7),these groups may acquire the positive charges making them detectable.

Negative charges: In some other embodiments, the negative charges cangenerally be induced on the reporter moiety through the incorporation ofalcohol hydroxyl and/or phenolic hydroxy functionalities on the aromaticor aliphatic skeleton. Given below are some of the proposed reportermoieties which meet the above mentioned criteria. The fragments listedbelow may be available and able to link to the linker through the aminoterminal. The additional advantage could be that the reagents that areproposed for the phosphoramidate linkage formation may be the same asthis amide linkage formation (Therefore reducing costs of the systemfurther).

Moreover, at least in part due to the stability of this linkage to thealkaline pH (above 7), the process of induction of negative charge wouldbe of no or substantially small interference.

For the purpose of illustration, the following three non-limitingexamples are presented. These examples are provided only for the purposeof illustration and therefore should not be considered to limit thescope of the invention. As such, any modifications on the followingexamples are certainly included in the scope of the invention. Forexample, any substitution of one or more groups (e.g. —OH, ═O, COOH, andothers) linked to the examples can be practiced. Also oligomerization orpolymerization of one of more of the following examples can also bepermitted. Further any other chemical structure or molecule with variousdimensions (e.g. length, size, and mass of the reporter moiety) can beused as a reporter moiety if such chemical structure or molecule isconfigured to provide a charged mass to the synthetic nucleotide andalso not to affect polymerization reaction of nucleotides partially orentirely.

After acquiring the charges, some of these reporters in certainembodiments may exist as follows,

Whereas, the reporter-1 and reporter-3 may be available on shelf,reporter-2 may be custom synthesized.

The reporter moieties proposed can generally (be) thus:

1. Easily available or synthesizable;

2. bear a large charge;

3. Not costly; and/or

4. easy to link and cleave.

Final compounds (monomers): Based on the above propositions, the finalstructures of the nucleotides along with the linkers and the reporterswould be as follows at least in certain parts of embodiments. Thefollowing examples of some final compounds are also provided for thepurpose of illustration and therefore should not be considered to limitthe scope of the invention. As described above, any variations permittedfor a nucleotide or its derivative, a linker and a high charge massreporter moiety are also permitted to a final compound. Thus, for theadenosine as a nucleotide at the 5′-phosphate terminal in some examples,if the linker is, say, C 13 equivalent (option 3 above), the variouslinkers would make the final structures looks as below:

One proposed final synthetic nucleotide-1 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

Another proposed synthetic nucleotide-2 (note the reporter is in monomerform and this can be increased by aggregating these monomers to increasecharge mass as required):

Still another proposed synthetic nucleotide-3 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

The following is a non-limiting, illustrative example of synthesisprotocols used in at least some embodiments:

-   -   1. Synthesis of 5′-phosphoramidates of Adenosine: (Linkage of        Nucleotide with the diamine linker). Method of Chu et all can be        used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamantes and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and was        incubated at room temperature with constant stirring. The        reaction was monitored till completion.    -   2. Synthesis of Final proposed structures: (Linkage of the        diamine linker with reporter moiety). Method of Chu et all can        be used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamines and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and        then incubated at room temperature with constant stirring. The        reaction was monitored till completion.

One advantage of the similar procedure is that it may work out for boththe steps leading to the formation of final compounds as monomers.

In some illustrative examples of some embodiments, (see below) cleavageof the linkers and reporter moieties may need to be done. The linkageslike phosphoramidates can generally be rather readily cleaved by the useof acids like Trifluoroacetic acid at an ambient temperature. By way ofan illustrative example, the proposed synthetic nucleotide-2demonstrated as a probable 3D view below. The aromatic ring at thebottom left of the molecule bears three hydroxy functions which couldpotentially get converted to the negative charge under slight alkalineconditions. Following is the 3D conformation of the Adenosine attachedwith the Reporter-1 through linker 3 and the related data.

Approximate distance between the phosphoramidate and terminal chargedatom may be about 20 angstroms, which could generally be sufficient toinduce the charge potential in the surface for detection. This distancecan further be altered with the further modifications in the phase atleast in part by changing the linker lengths. The charge on the terminalreporter moieties can also be changed by the variations in the chemistryof reporter moieties.

In one embodiment, the reporter composition recited in the appendedclaims comprises any nucleotide with a cleavable linker moleculeattached to a high charge mass moiety, wherein the synthetic nucleotide(otherwise referred to as the reporter composition) has a charge that issufficient to cause a detectable change in the property of a sensitivedetection nanostructure, when the reporter composition is operablycoupled to the nanostructure (as for example, by addition of thesynthetic nucleotide (reporter composition) to a nascent chain during asequencing by synthesis procedure).

EXAMPLES

The following description is an illustrative example of some embodimentsof the present disclosure.

Example 1 DNA Sequencing

The sequencing methodology in one example may not use fluorescence andexpensive sensitive cameras, but instead may detect the addition of thesynthetic nucleotides described in some aspects of the presentdisclosure, at least in part by sensing the electrical charge ofreporter moiety, using sensitive nanostructures that may be capable ofdetecting a build up of charge mass at, or near, their surface. When anew nucleotide is added to the growing polymer in a sequencing bysynthesis reaction, the charge density at, or near the surface of thesensitive nanostructure may increase and this can be detected by achange in property in the sensitive detection nanostructure (forinstance, if using a nanowire, or carbon nanotube, as the detectingstructure, an increase in charge caused by the addition of a nucleotideclose to its surface may be detected by a change in resistance in thewire, due to a phenomenon called the field effect). However, as thepolymer grows, the signal may diminish as the charges carried by thenucleotides being added may be too far away from the sensitivenanostructure (e.g. nanowire) to illicit a change in property of thesensitive detection nanostructure and no signal may be observed.Therefore, the ‘read length’ (amount of sequence data that is able to beobtained by this method of nucleotide sequencing) can be limited.

As used herein this particular example, a “sensitive detectionnanostructure” can be any structure (nanoscale or not) which can becapable of detecting any change in charge at, or near it's surface andat any point may have at least one cross-sectional dimension less thanabout 500 nanometers, typically less than about 200 nanometers, moretypically less than about 150 nanometers, still more typically less thanabout 100 nanometers, still more typically less than about 50nanometers, even more typically less than about 20 nanometers, stillmore typically less than about 10 nanometers, and even less than about 5nanometers. In other embodiments, at least on of the cross-sectionaldimensions can generally be less than about 2 nanometers, or about 1nanometer. In one set of embodiments the sensitive detectionnanostructure can have at least one cross-sectional dimension rangingfrom about 0.5 nanometers to about 200 nanometers.

The properties of a sensitive detection nanostructure may change inresponse to surface, or near surface charge in a way that may bemeasurable via piezoelectric measurements, electrochemical measurement,electromagnetic measurement, photodetection, mechanical, measurement,acoustic measurement, gravimetric measurement and the like. An exampleof a sensitive detection nanostructure may include, but not limited to,two dimension field effect transistors, a cantalevers, nanowires, carbonnanotubes, and all piezoelectric macro-, micro-, nano-, pico-, zempto-,or smaller structures.

Certain embodiments of the present disclosure may address thislimitation, at least in part by using synthetic nucleotides that maycomprise normal nucleotides, with a high negative (or positive) chargemass reporter moiety attached via a linker molecule (for instance,attached to the 5′ phosphate group), with the linker length increasingas the reaction progresses. This charge mass can be designed to ‘reachdown’ to the sensitive nanostructure (e.g. nanowire) to cause a changein property of the sensitive detection nanostructure (e.g. a fieldeffect or other piezo-electric change in the structure depending on thesensitive detection nanostructure used). To enable a good qualitycontrol measure and to ensure long read lengths by eliminating the buildup of many reporter moieties which would cause an ever increasing fieldeffect, these reporter moieties can be cleaved at least in certainembodiments, to allow for the addition of the next nucleotide in thesequencing by synthesis sequence.

Therefore, in some embodiments the cyclical reaction may comprise atleast some or whole of the following entire or partial series of events:

-   -   1. The template ssDNA molecule to be sequenced can be either        ligated to the sensitive detection nanostructure and a primer        added, bind to a pre-immobilized primer sequence on the        sensitive detection nanostructure, or uncoiled and elongated in        a microfluidics channel arrayed with sensitive detection        nanostructures.    -   2. The sensitive detection nanostructures can be washed with        water, or a low salt buffer (such as 1×SSC)    -   3. A measure of the sensitive detection nanostructure can be        made.    -   4. A mixture containing one synthetic nucleotide, the polymerase        and other elements required for the polymerization reaction can        be added. In one example, if the nucleotide added is        complimentary to the base on the minus strand immediately after        the primer sequence, it maybe incorporated into the growing        chain by the polymerase.    -   5. The reaction can then be washed with either water or a low        salt buffer (such as 1×SSC).    -   6. A measure of the sensitive detection nanostructure can be        made which can observe the effect caused by the high charge mass        of the reporter moiety.    -   7. The reporter moiety can then be cleaved (for instance by an        acid solution or enzymatically).    -   8. Points 2 through 7 can be repeated for each of the four        nucleotides. And this can be repeated repeatedly until a clear        signal may degrade.

For some embodiments wherein the template molecule is immobilized to, orbound to a probe that can be in turn immobilized to the sensitivedetection nanostructure, the linker lengths that attach the high chargereporter moiety to the synthetic nucleotides may increase to enable thecharge to ‘reach down’ to the sensitive detection nanostructure to exertan effect. This may be necessary at least in some embodiments as thegrowing nucleotide polymer may move the next nucleotide addition sitefarther and farther from the sensitive detection nanostructure as thesequencing by synthesis reaction may progress.

For some other embodiments wherein the template molecules is notimmobilized to the sensitive detection nanostructure, or hybridized to aprimer/probe that can be in turn immobilized to the sensitive detectionnanostructure, and can be instead free or immobilized horizontallyacross a cluster of sensitive detection nanostructures, a single linkerlength can be used for each of the cycle reactions.

Example 2 Primer Extension

In some embodiments, the synthetic nucleotides described in some aspectsof the present disclosure for primer extension experiment wherein thedetection is performed on electrical biosensors (nanowire/nanotube FETs,2D FETS, nanopores, piezo-electric films/surfaces, etc). Primerextension is generally defined as a technique that can map or determinea 5′ end of DNA or RNA. For example, primer extension can be used todetermine the start site of the transcription start site for a gene.This technique generally requires a labelled primer, which iscomplementary to a region near the 3′ end of the target gene. The primeris allowed to anneal to the transcript of the target gene and reversetranscriptase is used to synthesize complementary cDNA to the transcriptuntil it reaches the 5′ end of the transcript. By running the product ona polyacrylamide gel, it can be possible to determine thetranscriptional start site, as the length of the sequence on the gelrepresents the distance from the start site to the labelled primer.During the synthesis of cDNA, the synthetic nucleotides disclosed inthis application can be used and added to the nascent cDNA chain. Theaddition of the specific synthetic nucleic acid (e.g. deoxynucleotidewith Adenine, Guanine, Thymidine, or Cystine) can be detected by ananosensor. The nanosensor, which is further described below can beattached to the primer so that the nascent cDNA chain may be attached tothe nanosensor in some embodiments. Alternatively, in some otherembodiments, the transcript of the target sequence may be attached tothe nanosensor (e.g. nanowires, nanotubes, nanobeads, nanopores,nanogaps and others).

Biosensors

As used in various embodiments, a biosensor is generally a device forthe detection of an analyte that combines a biological component with aphysicochemical detector component. In some embodiments, it may comprisethree parts: 1. the sensitive biological element (biological material(eg. tissue, microorganisms, organelles, cell receptors, enzymes,antibodies, nucleic acids, etc), a biologically derived material orbiomimic). The sensitive elements can be created by biologicalengineering; 2. the transducer or the detector element (works in aphysicochemical way; optical, piezoelectric, electrochemical, etc.) thattransforms the signal resulting from the interaction of the analyte withthe biological element into another signal (i.e., transducers) that canbe more easily measured and quantified; 3. associated electronics orsignal processors that is primarily responsible for the display of theresults in a user-friendly way. In some other examples, the signalprocessing unit may further comprise one or more of a signal sensingunit, a signal recording unit, a data processing unit, and a datareporting unit.

Nanostructures

As used in various embodiments, a nanowire is an elongated nanoscalesemiconductor which, at any point along its length, has at least onecross-sectional dimension and, in some embodiments, two orthogonalcross-sectional dimensions less than 500 nanometers, preferably lessthan 200 nanometers, more preferably less than 150 nanometers, stillmore preferably less than 100 nanometers, even more preferably less than70, still more preferably less than 50 nanometers, even more preferablyless than 20 nanometers, still more preferably less than 10 nanometers,and even less than 5 nanometers. In other embodiments, thecross-sectional dimension can be less than 2 nanometers or 1 nanometer.In one set of embodiments the nanowire has at least one cross-sectionaldimension ranging from 0.5 nanometers to 200 nanometers. Where nanowiresare described having a core and an outer region, the above dimensionsrelate to those of the core. The cross-section of the elongatedsemiconductor may have any arbitrary shape, including, but not limitedto, circular, square, rectangular, elliptical and tubular. Regular andirregular shapes are included. A non-limiting list of examples ofmaterials from which nanowires of the invention can be made appearsbelow.

Nanotubes are a class of nanowires that may find use in the inventionand, in one embodiment, devices of the invention include wires of scalecommensurate with nanotubes. As used herein, a “nanotube” is a nanowirethat has a hollowed-out core, and includes those nanotubes know to thoseof ordinary skill in the art. A “non-nanotube nanowire” is any nanowirethat is not a nanotube. In one set of embodiments of the invention, anon-nanotube nanowire having an unmodified surface (not including anauxiliary reaction entity not inherent in the nanotube in theenvironment in which it is positioned) is used in any arrangement of theinvention described herein in which a nanowire or nanotube can be used.A “wire” refers to any material having a conductivity at least that of asemiconductor or metal. For example, the term “electrically conductive”or a “conductor” or an “electrical conductor” when used with referenceto a “conducting” wire or a nanowire refers to the ability of that wireto pass charge through itself. Preferred electrically conductivematerials have a resistivity lower than about 10⁻³, more preferablylower than about 10⁻⁴, and most preferably lower than about 10⁻⁶ or 10⁻⁷ohm-meters.

Nanopore generally has one or more small holes in an electricallyinsulating membrane that can be used as a single-molecule detector. Insome cases, it can be a biological protein channel in a high electricalresistance lipid bilayer or a pore in a solid-state membrane. Nanoporeis generally a spherical structure in a nanoscale size with one or morepores therein. According to some aspects, a nanopore is made of carbonor any conducting material.

Nanobead is generally a spherical structure in a nanoscale size. Theshape of nanobead is generally spherical but can also be circular,square, rectangular, elliptical and tubular. Regular and irregularshapes are included. In some examples, the nanobead may have a poreinside.

Nanogap is generally used in a biosensor that consists of separationbetween two contacts in the nanometer range. It senses when a targetmolecule, or a number of target molecules hybridize or binds between thetwo contacts allowing for the electrical signal to be transmittedthrough the molecules.

The foregoing nanostructures, namely, nanowire, nanotube, nanopore,nanobead, and nanogap are described to provide the instant illustrationof some embodiments, and not for limiting the scope of the presentinvention. In addition to the foregoing examples, any nanostructure thathas a nanoscale size and is suitable to be applied to nucleic aciddetection methods and apparatus as disclosed in the application shouldalso be considered to be included in the scope of the invention.

In general, sensing strategies for use with nanostructures ornanosensors to detect molecules and compounds is to sense changes in thecharge at, or near their surfaces, or across a nanogap or nanopore,which cause a measurable change in their properties (such as fieldeffect transistors, nanogaps, or piezoelectric nanosensors) to detect &quantify target nucleic acids (DNA, RNA, cDNA, etc).

Aspects of the invention provide a nanowire or nanowires preferablyforming part of a system constructed and arranged to determine ananalyte in a sample to which the nanowire(s) is exposed. “Determine”, inthis context, means to determine the quantity and/or presence of theanalyte in the sample. Presence of the analyte can be determined bydetermining a change in a characteristic in the nanowire, typically anelectrical characteristic or an optical characteristic. E.g. an analytecauses a detectable change in electrical conductivity of the nanowire oroptical properties. In one embodiment, the nanowire includes,inherently, the ability to determine the analyte. The nanowire may befunctionalized, i.e. comprising surface functional moieties, to whichthe analytes binds and induces a measurable property change to thenanowire. The binding events can be specific or non-specific. Thefunctional moieties may include simple groups, selected from the groupsincluding, but not limited to, —OH, —CHO, —COOH, —SO₃H, —CN, —NH₂, —SH,—COSH, COOK, halide; biomolecular entities including, but not limitedto, amino acids, proteins, sugars, DNA, antibodies, antigens, andenzymes; grafted polymer chains with chain length less than the diameterof the nanowire core, selected from a group of polymers including, butnot limited to, polyamide, polyester, polyimide, polyacrylic; a thincoating covering the surface of the nanowire core, including, but notlimited to, the following groups of materials: metals, semiconductors,and insulators, which may be a metallic element, an oxide, an sulfide, anitride, a selenide, a polymer and a polymer gel. In another embodiment,the invention provides a nanowire and a reaction entity with which theanalyte interacts, positioned in relation to the nanowire such that theanalyte can be determined by determining a change in a characteristic ofthe nanowire.

Field Effect Transistor (FET)

Field effect generally refers to an experimentally observable effectsymbolized by F (on reaction rates, etc.) of intramolecular coulombicinteraction between the centre of interest and a remote unipole ordipole, by direct action through space rather than through bonds. Themagnitude of the field effect (or ‘direct effect’) may depend on theunipolar charge/dipole moment, orientation of dipole, shortest distancebetween the centre of interest and the remote unipole or dipole, and onthe effective dielectric constant. This is exploited in transistors forcomputers and more recently in DNA field-effect transistors used asnanosensors.

Field-effect transistor (FET) is generally a field-effect transistor,which may use the field-effect due to the partial charges ofbiomolecules to function as a biosensor. The structure of FETs can besimilar to that of metal-oxide-semiconductor field-effect transistor(MOSFETs) with the exception of the gate structure which, in biosensorFETs, may be replaced by a layer of immobilized probe molecules whichact as surface receptors. When target biomolecules hybridize or bind, tothe receptors, the charge distribution near the surface changes, whichin turn modulates current transport through the semiconductor transducer(e.g. nanowire).

Biological Samples

The term sample or biological sample generally refers to any cell,tissue, or fluid from a biological source (a “biological sample”), orany other medium, biological or non-biological, that can be evaluated inaccordance with the invention including, such as serum or water. Asample includes, but is not limited to, a biological sample drawn froman organism (e.g. a human, a non-human mammal, an invertebrate, a plant,a fungus, an algae, a bacteria, a virus, etc.), a sample drawn from fooddesigned for human consumption, a sample including food designed foranimal consumption such as livestock feed, milk, an organ donationsample, a sample of blood destined for a blood supply, a sample from awater supply, or the like. One example of a sample is a sample drawnfrom a human or animal to determine the presence or absence of aspecific nucleic acid sequence.

Nucleic Acid or Oligonucleotide

The terms nucleic acid or oligonucleotide or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 131:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240. 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994).Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995). Chem. Soc. Rev. pp. 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

Sensing Strategies

In one aspect of the invention, a biological material configured to binda nanostructure is a nucleic acids. Such nucleic acids may include DNA,RNA, and any derivatives thereof. In one embodiment, the biologicalmaterial is DNA. When DNA is attached to the nanostructure, the numberof nucleotides may range from 5 bases to 100 bases. In some embodiments,the number of DNA nucleotides may be 7 bases, 10 bases, 15 bases, 20,bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 60bases, 70 bases, 80 bases, 90 bases and 100 bases. In some otherembodiments, ribonucleic acids and any nucleic acid derivatives may beattached to the nanostructure. In still some other embodiments, DNA, RNAand its derivatives may be used simultaneously. Therefore in oneexample, DNA sequences may be attached to the nanostructure, whereas inanother example, RNA sequences may be attached to the nanostructure,still another example, nucleic acid derivatives such as adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof may be attached to the nanostructure. In some otherexamples, nucleotide sequences comprising DNA and RNA, DNA and nucleicacid derivatives, RNA and derivatives, and DNA, RNA and derivatives maybe attached to the nanostructure.

In another aspect of the invention, a nanostructure is conducting andcan sense the electric charge, which is originated from the syntheticnucleotide as disclosed in this application, at its surface, vicinity,inner tubes and/or the pores therein. One of key aspects of anydiagnostic device is the ability to perform accurate detection ofbiomolecules with the performance determined by how well it detectsspecifically (i.e. a low false positive rate) and sensitively (i.e. alow false negative rate). Nanosensors that can sense changes in thecharge at, or near their surfaces, or across a nanogap or nanopore,which cause a measurable change in their properties, at least in partdue to the target molecule binding to a probe immobilized on or near thenanostructures, provide a method for ultra-sensitive detection withoutor with limited use of the need for labels (expensive chemicals that canbe bound to the biomolecule or molecule of interest to enable detectiondevices to ‘sense’ them).

The present disclosure generally relates to molecular biologicalprotocols and sensing strategies for use with nanosensors that maydetect molecules and compounds by sensing changes in the charge at, ornear their surfaces, or across a nanogap or nanopore, which cause ameasurable change in their properties (such as field effect transistors,nanogaps, or piezoelectric nanostructures or nanosensors) to detect &quantify target nucleic acids (DNA. RNA, cDNA, etc). The basic functionof these biosensors may require that a nucleotide polymer probe (orsynthetic nucleotide polymer such as PNA, Morpholinos, etc) beimmobilized on, or near to, the nanostructures and the build up oftarget molecules binding to the probe can cause an increase in chargedensity at or near the surface of the nanostructures or nanosensors, dueto the charge of the probe. For instance, an amplified PCR fragmentbinding to a probe (with a reverse complimentary sequence to the targetnucleotide polymer), immobilized on a nanowire can cause a measurablechange conductance (ΔG) due to the increase in negative charge at, ornear to the nanowire's surface, due to a phenomena called the fieldeffect. In some embodiments, the electric charge present in thesynthetic nucleotide, which is originated from the nucleotide itself andthe high charge mass reporter moiety, can be detected by thenanostructures/nanosensors.

These nanosensors may offer the potential for sensitive and dynamicdetection of biomolecules, however, this sensitivity may bring with it anumber of issues. For instance, natural fluctuations of charge at thesurface, within the sample matrix may cause noise, in part due to theflanking sequences of target nucleotide polymer molecules (i.e. the overhanging sequences that don't bind to the probes). Furthermore, if manytarget molecules are being detected at the same time on an array ofnanosensors, it would be favorable to standardize the size and thereforecharge mass, of each of these molecules to allow for more stringentcomparisons and quality control. Moreover, having a standard size forall target molecules allows for standardization of probe hybridizationconditions in the array assay design.

Biosensor System

Biosensor is generally an analytical device that may convert molecularevents into electrical signals. The nanostructures used in a biosensorare generally used to detect components of interest such as nucleicacids. Biosensors can generally operate in the liquid or gas phase,opening up an enormous variety of applications, e.g., for integrateddevices and for downstream applications. Therefore, the biosensors canbe manufactured inexpensively and portable and are optionally used asimplantable detection and monitoring devices. Alternatively, thebiosensor can be coupled with other high-resolution apparatus such asmass-spectroscopy and provide further information including thedetection of presence, abundance and/or structural variation of thetarget biomolecules.

One aspect of the invention involves a sensing element of a biosensor,which can be an electronic sensing element, and a nanowire able todetect the presence, or absence, of an analyte in a sample (e.g. a fluidsample) containing, or suspected of containing, the analyte. Nanoscalesensors of the invention may be used, for example, in chemicalapplications to detect pH or the presence of metal ions; in biologicalapplications to detect a protein, nucleic acid (e.g. DNA, RNA, etc.), asugar or carbohydrate, and/or metal ions; and in environmentalapplications to detect pH, metal ions, or other analytes of interest.

Another aspect of the invention involves an article of a biosensorcomprising a sample exposure region and a nanowire able to detect thepresence of absence of an analyte. The sample exposure region may be anyregion in close proximity to the nanowire wherein a sample in the sampleexposure region addresses at least a portion of the nanowire. Examplesof sample exposure regions include, but are not limited to, a well, achannel, a microchannel, and a gel. In preferred embodiments, the sampleexposure region holds a sample proximate the nanowire, or may direct asample toward the nanowire for determination of an analyte in thesample. The nanowire may be positioned adjacent to or within the sampleexposure region. Alternatively, the nanowire may be a probe that isinserted into a fluid or fluid flow path. The nanowire probe may alsocomprise a micro-needle and the sample exposure region may beaddressable by a biological sample. In this arrangement, a device thatis constructed and arranged for insertion of a micro-needle probe into abiological sample will include a region surrounding the micro-needlethat defines the sample exposure region, and a sample in the sampleexposure region is addressable by the nanowire, and vice-versa. Fluidflow channels can be created at a size and scale advantageous for use inthe invention (microchannels) using a variety of techniques such asthose described in International Patent Publication No. WO 97/33737,published Sep. 18, 1997, and incorporated herein by reference.

In another aspect of the invention, an article may comprise a pluralityof nanowires able to detect the presence or absence of a plurality ofone or more analytes. The individual nanowires may be differentiallydoped as described above, thereby varying the sensitivity of eachnanowire to the analyte. Alternatively, individual nanowires may beselected based on their ability to interact with specific analytes,thereby allowing the detection of a variety of analytes. The pluralityof nanowires may be randomly oriented or parallel to one another.Alternatively, the plurality of nanowires may be oriented in an array ona substrate.

Where a detector is present, any detector capable of determining aproperty associated with the nanowire can be used. The property can beelectronic, optical, or the like. An electronic property of the nanowirecan be, for example, its conductivity, resistivity, etc. An opticalproperty associated with the nanowire can include its emissionintensity, or emission wavelength where the nanowire is an emissivenanowire where emission occurs at a p-n junction. For example, thedetector can be constructed for measuring a change in an electronic ormagnetic property (e.g. voltage, current, conductivity, resistance,impedance, inductance, charge, etc.) can be used. The detector typicallyincludes a power source and a voltmeter or amp meter. In one embodiment,a conductance less than 1 nS can be detected. In a preferred embodiment,a conductance in the range of thousandths of a nS can be detected. Theconcentration of a species, or analyte, may be detected from less thanmicromolar to molar concentrations and above. By using nanowires withknown detectors, sensitivity can be extended to a single molecule. Inone embodiment, an article of the invention is capable of delivering astimulus to the nanowire and the detector is constructed and arranged todetermine a signal resulting from the stimulus. For example, a nanowireincluding a p-n junction can be delivered a stimulus (electroniccurrent), where the detector is constructed and arranged to determine asignal (electromagnetic radiation) resulting from the stimulus. In suchan arrangement, interaction of an analyte with the nanowire, or with areaction entity positioned proximate the nanowire, can affect the signalin a detectable manner. In another example, where the reaction entity isa quantum dot, the quantum dot may be constructed to receiveelectromagnetic radiation of one wavelength and emit electromagneticradiation of a different wavelength. Where the stimulus iselectromagnetic radiation, it can be affected by interaction with ananalyte, and the detector can detect a change in a signal resultingtherefrom. Examples of stimuli include a constant current/voltage; analternating voltage, and electromagnetic radiation such as light.

Another aspect of the present invention provides an article comprising ananowire and a detector constructed and arranged to determine a changein an electrical property of the nanowire. At least a portion of thenanowire is addressable by a sample containing, or suspected ofcontaining, an analyte. The phrase “addressable by a fluid” is definedas the ability of the fluid to be positioned relative to the nanowire sothat an analyte suspected of being in the fluid is able to interact withthe nanowire. The fluid may be proximate to or in contact with thenanowire.

In some embodiments, the nanostructures can be assembled into aplurality of parallel arrays such as micro-columns at higher densitiesthan is and in a format compatible with currently availablemicro-fluidic systems. The nanostructure arrays optionally comprise aplurality of nanostructures such as nanowires, nanotubes, nanopores,nanobeads, nanogaps, or a combination thereof. Each nanostructure of thearray can be electrically connected, e.g., via two or more electrodes toa battery for applying a voltage across the nanowire and a detector, fordetection of any changes in conductance of the nanowire. Alternatively,each nanostructure separately receives electricity or only a portion ofnanostructures arrayed together may be electrically connected.

A single detector or a combination of detectors is optionally used todetect the signal from the array of nanowires. For example, eachnanowire linked to a probe comprising different target sequence, whichmay be bound to a same or different probe, is optionally detectedseparately, such that a spatial array of a plurality of nanowires can beused to quickly identify, e.g., a plurality of different nucleotidesequences present in a biological sample such as blood. In someexamples, a plurality of patches of nanostructures are prepared in thearray and each patch presents different probes to detect multiple targetsequences in a biological sample. Alternatively, in some other examples,an entire nanostructures present in the array may present same probes,thereby only one target sequence would be tested for its presence,abundance and/or variation in the sequence.

The detection by the nanostructure or nanosensor is generally a changein conductance of the nanostructure or of its environment. The signalcan be expressed in terms of a change in the voltage across thenanostructure, or the current through the nanostructure. Such changesare typically detected electrically, e.g., with a voltmeter and/or acurrent meter. Alternatively, the signal is detected digitally. In oneembodiment, a voltage is applied across a nanostructure, e.g., ananowire, providing a steady state signal. When a binding event occurson the probe attached to the nanostructure, the electric field in thevicinity of the nanostructure changes and the conductance of thenanostructure changes, producing a fluctuation or shift in the steadystate signal. The signal may be detected, electrically or digitally, andprovides real time detection of the event of interest.

Biosensor can also be integrated into a system for detecting a presence,level and/or variation of biomolecules. In one aspect, such system mayinclude an electrical power supply, monitoring system for applying andmeasuring electrical current across the nanostructure element. Inanother aspect, such system may further include data processingcapabilities to enable the programmed operation of the nanostructuresand to receive, store, and provide useful analysis and display of thedata that is obtained. In addition a computer system to process theobtained data as well as additional processor(s) may be integrated intoa biosensor system if desired. The computer system or any additionalelements present in a biosensor system may provide a software(s) foranalyzing the data or for automatic operation and/or manual(s) toperform detection processes with a biosensor. Furthermore, anyadditional elements that may enhance the performance of a biosensorsystem can be added. A biosensor of the present invention can collectreal time data.

Example 3 Hybridization such as Microarrays

In still some other embodiments, the synthetic nucleotide disclosed insome aspects of the present disclosure can be used for hybridizationprocedures. Some non-limiting, illustrative examples of thehybridization procedures include a microarray for nucleonic acids aswell as proteins. Further any other procedures that need hybridizationand can determine presence, abundance or any structural variation of thetarget biomolecules can be included.

In one example, the probes used in a microarray can be attached to amedium such as nanosensors (e.g. nanowires, nanotubes, nanobeads,nanaopores, nanogaps, and other nanostructures). In some cases, thetarget nucleotide sequences obtained from the biological samples wouldbe labeled and contacted with the probes. In such cases, the targetnucleotide may incorporate the synthetic nucleotides thereby beinglabeled with “high charge mass”. As such, the binding of the targetsequences to the probes can be readily determined by the nanosensorsthat the probes are attached to. Moreover, the synthetic nucleotidedisclosed in this application can be used, for example, in the methodsof detecting presence, abundance and/or structural variation of nucleicacids as disclosed in the related application of the subjectapplication, U.S. provisional application No. 61/094017 filed on Sep. 3,2008, the disclosures of which are hereby expressly incorporated byreference in their entirety and are hereby expressly made a portion ofthis application. As such, the use of the synthetic nucleotide of thisapplication is not limited and can be further extended if applicable.

1. A reporter composition, comprising: a nucleotide or its derivative; alinker molecule, wherein the linker molecule is attached to thenucleotide or its derivative and a high charge mass moiety; and a highcharge mass moiety, wherein the high charge mass moiety comprises acharge mass that is sufficient to change a property of a sensitivedetection nanostructure operably coupled to the reporter composition. 2.The reporter composition of claim 1, wherein the nucleotide or itsderivative is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.
 3. The reporter composition of claim 1, wherein thelinker molecule comprises a molecule of the following general formula:H₂N-L-NH₂ wherein L comprises a linear or branched chain comprising analkyl group, an oxy alkyl group, or the combination thereof.
 4. Thereporter composition of claim 3, wherein L comprises a linear chaincomprising an alkyl group, an oxy alkyl group, or the combinationthereof.
 5. The reporter composition of claim 4, wherein a number of thealkyl group, the oxy alkyl group, or the combination thereof in thelinear chain is 1 to
 100. 6. The reporter composition of claim 5,wherein the number of the alkyl group, the oxy alkyl group, or thecombination thereof in the linear chain is 1 to
 75. 7. The reportercomposition of claim 5, wherein the number of the alkyl group, the oxyalkyl group, or the combination thereof in the linear chain is 1 to 50.8. The reporter composition of claim 5, wherein the number of the alkylgroup, the oxy alkyl group, or the combination thereof in the linearchain is 1 to
 25. 9. The reporter composition of claim 5, a number ofthe alkyl group, the oxy alkyl group, or the combination thereof in thelinear chain is 1 to
 1000. 10. The reporter composition of claim 5, anumber of the alkyl group, the oxy alkyl group, or the combinationthereof in the linear chain is more than
 1000. 11. The reportercomposition of claim 1, wherein the linker molecule is configured not toaffect nucleotide polymerization by a polymerase.
 12. The reportercomposition of claim 1, wherein a net charge mass of the high chargemass moiety is positive or negative.
 13. The reporter composition ofclaim 1, wherein a net charge mass of the high charge mass moiety isvariable depending on pH.
 14. The reporter composition of claim 1,wherein the high charge mass moiety comprises an aromatic and/oraliphatic skeleton, wherein said skeleton comprises one or more of atertiary amino group, an alcohol hydroxyl group, a phenolic hydroxygroup, and any combinations thereof.
 15. The reporter composition ofclaim 1, wherein the high charge mass moiety comprises one or more ofthe following groups, and derivatives and combinations thereof:


16. The reporter composition of claim 15, wherein a number of thegroups, any derivatives thereof, and any combinations thereof in thehigh charge mass moiety is 1 to
 10. 17. The reporter composition ofclaim 15, wherein a number of the groups, any derivatives thereof, andany combinations thereof in the high charge mass moiety is 11 to
 50. 18.The reporter composition of claim 15, wherein a number of the groups,any derivatives thereof, and any combinations thereof in the high chargemass moiety is 51 to
 100. 19. The reporter composition of claim 15,wherein a number of the groups, any derivatives thereof, and anycombinations thereof in the high charge mass moiety is more than 100.20. The reporter composition of claim 1, wherein the high charge massmoiety is configured not to affect nucleotide polymerization by apolymerase.
 21. The reporter composition of claim 1, wherein the linkermolecule and/or the high charge mass moiety is configured to beremovable.
 22. A reporter composition, comprising:

wherein R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.
 23. A reporter composition, comprising:

wherein R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.
 24. A reporter composition, comprising:

wherein R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.
 25. A kit for determining a nucleotide sequence,comprising the reporter composition of any of claim 1, 22, 23 or
 24. 26.A method of synthesizing the reporter composition of claim 1,comprising: generating a first covalent linkage between the nucleotideor its derivative and a first amine group of the linker, wherein aphosphate group, a sugar or a base of the nucleotide or its derivativeis linked to the first amine group of the linker; and generating asecond covalent linkage between a second amine group of the linker andany functional group present in the high charge mass moiety: wherein thelinker comprises at least two amine groups.
 27. The method of claim 26,wherein the nucleotide or its derivative comprises a monophosphategroup.
 28. The method of claim 26, wherein the nucleotide or itsderivative is selected from the group consisting of adenosinemonophosphate (AMP), guanosine monophosphate (GMP), cytidinemonophosphate (CMP), thymidine monophosphate (TMP), and uridinemonophosphate (UMP).